It is increasingly being recognized that damage to the brain's circadian timing system contributes to the disability in patients with a variety of neurodegenerative disorders, including Parkinson's and Huntington's disease, and progressive supranuclear palsy, by disrupting wake-sleep and activity cycles, and increasing cognitive impairment. In this proposal we will study the brain circuitry that underlies circadian timing of wake-sleep, feeding, activity, body temperature, and secretion of melatonin and corticosteroids. The suprachiasmatic nucleus (SCN) is recognized as the brain's master biological clock, but the downstream circuitry that regulates these cycles is only now coming under study. SCN inputs to the subparaventricular zone (SPZ) and dorsomedial nucleus of the hypothalamus (DMH) have been implicated in this process, but the neurotransmitters and connections involved for the most part remain unknown. We propose three aims to study the role played by GABAergic transmission in the SCN, SPZ, and DMH, and glutamatergic neurons in the DMH, in this process. We will use mice with loxP sites flanking the second exon in either the vesicular GABA transporter (Vgat) or the vesicular glutamate transporter type 2 (Vglut2), and will inject an adeno-associated viral vector containing the genes for Cre recombinase and green fluorescent protein (GFP) into either the SCN (Aim 1), the ventral or dorsal SPZ (Aim 2), or the DMH (Aim 3) to specifically delete Vgat expression (and therefore GABA neurotransmission) by neurons in the SCN, SPZ, or DMH, or Vglut2 expression (and therefore glutamatergic neurotransmission) by neurons in the DMH. We will measure the circadian cycles of locomotor activity, body temperature, feeding, wake-sleep, and secretion of melatonin and corticosterone, and determine the extent to which these depend upon either GABAergic transmission from the SCN, SPZ, or DMH, or glutamatergic transmission from the DMH. We will also use the GFP tracing from the injection sites, in conjunction with retrograde tracing from the targets and either Vgat or Vglut2 in situ hybridization, to determine the role played by glutamate and GABA as neurotransmitters from the circadian timing system to targets involved with wake-sleep, feeding, and hormone secretion. This information will identify key components and pathways, and roles of GABA and glutamate in the central circadian signaling system. PUBLIC HEALTH RELEVANCE: The circadian timing system in the brain regulates cycles of wake-sleep, cognitive function, feeding, and hormone secretion, but this system is damaged by many neurodegenerative disorders, and may contribute significantly to the behavioral disruption that is observed. In particular, disruptive behavior at night is one of the key reasons for nursing home placement. We will identify key brain circuitry that underlies circadian cycles of these important processes, a necessary first step toward controlling them and reducing the dysfunction experienced by patients with disorders such as Parkinson's and Huntington's disease.